Cancer cells found in circulation are believed to disseminate from tumors and form secondary sites. These malignant cells, termed circulating tumor cells (CTCs), may provide a vital parameter for cancer detection, staging, and developing treatment for tumor metastasis. However, these cells occur within the body at extremely low frequencies (1-500 CTCs/mL blood) and exist among billions of other blood cells (e.g., red blood cells, RBCs, and white blood cells, WBCs), which has motivated methods to achieve efficient isolation for subsequent analysis by targeting specific biomarkers. Larger cell size (>15 μm) compared to normal blood cells, for example, is a characteristic of certain cancers (e.g., non-small cell lung cancer, NSCLC) that allows specific targeting of CTCs from contaminating blood components. The standard mode of analysis is detection with immunostaining (e.g., DAPI positive, EpCAM positive, Cytokeratin positive, and CD45 negative) and enumeration, where a high EpCAM+ CK+ CD45− count has been found to correlate with disease progression and can be used to monitor treatment efficacy. However, the presence of CK+ cells in patients with benign disease and the intra and inter-tumor heterogeneity of both EpCAM and cytokeratin expression indicates the need to analyze other parameters that may correlate more accurately with malignancy.
Traditional methods for cell analysis and counting involve manual measurements, including hemocytometers to determine sample concentrations and microscope graticules for cell size measurements. These processes have become more automated with the discovery of the Coulter principle, which involves detecting events of electrical impedance changes and subsequent correlation to total count and particle size. This is the basis of the Coulter Counter, which is a current standard for cell enumeration and size measurements in laboratory settings. However, the Coulter Counter has a limited window of specificity, which makes it inaccurate in detecting the low frequency of these potentially malignant cells. Additionally, the complex nature of biofluids, which contain a high level of background components, makes it difficult to detect these rare cells of interest. The Coulter Principle also enabled flow cytometry, which is a gold standard for cellular analysis in clinical practice and basic science research. These systems can enumerate and measure cell morphological properties as well as molecular characteristics but are limited by their complexity and high-cost. As with the Coulter Counter, the scarcity of these target cells limits the use of traditional flow cytometers, which require at least tens of thousands of cells per sample and frequencies above ˜1% to surpass intrinsic noise.
Immunostaining methods can be used for identifying and analyzing rare cells from bodily fluids. Isolated cell samples are manually manipulated with a series of wash, labeling, and incubation steps to discern captured CTCs from contaminating blood components. Standardized stains such as DAPI (targeting DNA), CD-45 (targeting leukocytes), and cytokeratin (targeting epithelial cells) are used for analysis with fluorescence microscopy. Cells that have a DAPI and cytokeratin signal are defined as CTCs and are differentiated from white blood cells, which have a DAPI and CD-45 signal. Immunostaining and fluorescence microscopy enable detection and enumeration, but require time-consuming protocols to be performed by trained technicians as well as antibodies, which are often expensive and introduce significant variability in performance. These techniques are mostly used because the purity of CTC samples can be low, and allow improved classification accuracy. Additionally, these protocols may require red blood cell lysis, fixation, permeabilization and antibody binding reactions, which alter the native state of the cell population and limit the ability to perform further assays. Further, the label-based method is also limited by inter and intra-tumor heterogeneity, as cells within a single tumor and between different tumor types have different expression levels of these markers.